The modulation of RNA structure is an essential regulatory process in many cellular events, such as, for example, pre-mRNA splicing, assembly of spliceosomes, assembly of ribosomes, protein translation, which can be summarized under the generic term xe2x80x9cregulation of gene expression at the RNA levelxe2x80x9d. The so-called xe2x80x9cDEAD boxxe2x80x9d protein family of putative RNA helicases, named after the characteristic amino acid motif Asp-Glu-Ala-Asp (in the single-letter code DEAD), in this context plays a key part (in particular for the modulation of the secondary and tertiary structure of mRNA. DEAD box proteins are also involved in processing of DNA. The members of this family and some subfamilies have differences in their specific function and cellular localization. However, in addition to characteristic sequence homologies certain members also show similar biochemical properties (F. V. Fuller-Pace, Trends in Cell Biology, Vol 4, 1994, 271-274). The characteristic protein sequences of the DEAD proteins are highly conserved in evolution (S. R. Schmid and P. Lindner, Molecular and Cellular Biology, Vol 11, 1991, 3463-3471). Members of this protein family are found in various viruses, bacteria, yeasts, insects, molluscs and lower vertebrates up to mammals and are responsible for a large number of cellular functions. The fact that even relatively simple organisms such as, for example, the yeast Saccharomyces cerevisiae express numerous proteins of the DEAD box protein family and their subfamilies, suggests that each of these proteins contributes to the specific interaction with certain RNAs or RNA families (I. lost and M. Dreyfus, Nature Vol 372, 1994, 193-196). It has been shown that translation factors, such as eIF-4A and the proteins involved in the pre-mRNA splicing process, recognize specific RNA target sequences or structures. Nevertheless, to date there is little information about the structure and the synthesis of characteristic RNA sequences which require the DEAD proteins for recognition and for ATPase/RNA helicase reaction (A. Pause and N. Sonenberg, Current Opinion in Structural Biology Vol 3, 1993, 953-959).
The DEAD box protein family is an enzyme class which is growing and which is involved in the various reactions in post transcriptional regulation of gene expression. Because of the high number of different cellular DEAD box proteins, it is to be expected that specific RNA helicases are assigned to certain classes of gene products, e.g. viral proteins, heat shock proteins, antibody and MHC proteins, receptors, RNAs etc. This specificity indicates that members of this protein family are attractive pharmacological targets for active compound development.
Two of the subclasses of the DEAD box protein family are the DEAH proteins (having one specific amino acid replacement) and the DEXH protein (having two amino acid replacements in the main motif, X being any desired amino acid) families, which also play a part in the replication, recombination, repair and expression of DNA and RNA genomes (Gorbalenya, A. E., Koonin, E. V., Dochenko, A. P., Blinov, V. M., 1989: Nucleic Acids Res. 17, 4713-4729). The DEAD box proteins and their subfamilies are often designated xe2x80x9chelicase superfamily IIxe2x80x9d (Koonin, E. V., Gorbalenya, A. E., 1992: FEBS 298, 6-8). This superfamily has seven highly conserved regions. Altogether, up to now over 70 members belong to this superfamily II.
The following schematic representation of the DEAD family and the DEAH and DEXH families subfamilies (Schmid, S. R., Lindner P., 1991: Molecular and Cellular Biology 11, 3463-3471) shows the similarity between the families. The structure of eIF-4A, a member of a DEAD box protein, is also shown. The numbers between these regions show the distances in amino acids (AA). X is any desired, AA. Where known, functions have been assigned to the ranges. 
The ATPase motif (AXXXXGKT) is an amino-terminal conserved region and occurs in most proteins which bind nucleotides, i.e. also in other proteins which interact with DNA and RNA, such an DNAB (part of the primosome), UvrD (endonuclease), elongation factor 1 and transcription termination factor Rho (Ford M. J., Anton, I. A., Lane, D. P., 1988: Nature 332, 736-738). As used in this specification xe2x80x9cATPase activityxe2x80x9d is used to mean the ability to catalyze hydrolysis of ATP. The ATPase A and ATPase B motifs function together in the enzymatic process of ATP hydrolysis.
The second conserved region is the so-called DEAD box, or DEAH, DEXH or DEXX box in other families of the helicases and nucleic acid-dependent ATPases. This region represents the ATPase B motif. In the reaction mechanism, the N-terminal aspartic acid in the DEAD box binds Mg2+ via a water molecule (Pai, E. F., Krengel, U., Petsko, G. A., Gody, R. S., Katsch, W., Wittinghofer, A., 1990: EMBO J. 9, 2351-2359). Mg2+ in turn forms a complex with the xcex2- and gamma-phosphate of the nucleotide and is essential for the ATPase activity. Substitutions of the first two amino acids of the DEAD region in eIF-4A prevent ATP hydrolysis and RNA helicase activity, but not ATP binding (Pause, A., Sonenberg, N., 1992: EMBO J. 11, 2643-2654). The DEAD region additionally couples RNA helicase activity to ATPase activity. The hydrolysis of ATP provides the energy needed for RNA unwinding during helicase activity.
The third region investigated is the SAT region (sometimes also TAT). As a result of mutation in this region, RNA helicase activity is suppressed, but other biochemical properties are retained (Pause A. and Sonenberg N., 1992). As used in this specification xe2x80x9chelicase activityxe2x80x9d is used to mean the ability to directly or indirectly catalyze the unwinding of RNA.
The farthest carboxy-terminal region is the HRIGRXXR region, which is necessary for RNA binding and ATP hydrolysis.
As stated above, members of the DEAD box protein family bind ATP and nucleic acid. As used in this specification a protein that xe2x80x9cbinds nucleic acidxe2x80x9d is defined an a protein that forms complexes with nucleic acid. The binding can be measured by standard methods like Electrophoretic Mobility Shift Assay (EMSA) or ELISA, which are well known in the art. The following assays may also be used: Scintillation Proximity Assay (SPA, Amersham International, Little Chalfont, Buckinghamshire, England) and BIAcore (Biomolecule Interaction Analysis, Pharmacia, Upsala Sweden).
As used in this specification, a protein that xe2x80x9cbinds ATPxe2x80x9d is defined as a protein that will bind ATP as measured using an assay that measures ability of labeled ATP to bind to protein. The ATP may be labeled using radioactive or fluorescent label. One example of an ATP binding assay is described in Pause, et al. EMBO J. 11:2643 (1992), which is hereby incorporated by reference. Briefly, a protein according to the invention is incubated in a crosslinking reaction mixture containing Tris-HCl (pH 7.5), Mg acetate, 32P-ATP, glycerol and DTT in the presence or absence of poly(u) (Pharmacia) under a 15 watt germicidal lamp at 4xc2x0 C. Unlabelled ATP is then added, followed by addition of RNase A at 37xc2x0 C. Samples are boiled in SDS-PAGE sample buffer and electrophoresed.
It follows from the above-mentioned relationships that specific RNA helicases are attractive targets for pharmaceutically active substances. For example, it is also known that certain pathogenic viruses, which can cause diseases in humans, animals or plants, carry in their genome a gene encoding an RNA helicase, which is needed for accurate replication (E. V. Koonin, 1991). Thus, specific substances that interfere with, or modulate, the activity of such virus-specific helicases could be used to treat virally-mediated diseases. Because helicases are also found in plants, substances that modulate plant helicases may be used to protect plants against pathogenic viruses. (F. V. Fuller-Pace, Trends in Cell Biology, Vol. 4, 1994, 271-274). Helicases also make attractive targets for development of therapeutic treatments for various types of diseases. For example, hereditary diseases such as Werner""s syndrome and Bloom""s syndrome have been linked to the production of proteins with helicase structure. See Yu, et al. Science 272: 258 (1996) and Research News, Science 272: 193 (1996)(Werner""s); Ellis, et al. Cell 83:655 (1995), and D. Bassett xe2x80x9cGenes of Medical Interestxe2x80x9d In http://www.ncbi.nih.gov/xREFdb/ (Bloom""s). A nucleolar RNA helicase is recognized by the autoimmune antibodies from a patient with watermelon stomach. Valdez, et al., Nucl. Acid. Res., 24:1220 (1996). In retinoblastoma cancer cells, expression of a DEAD box protein is amplified. Godbout, et al. Proc. Natl. Acad. Sci. USA 90:7578 (1993). In addition, RNA processing plays a role in a number of processes that are implicated in other disease states. For example, in diabetic mice, the leptin receptor is abnormally spliced. Lee, et al. Nature 379:632 (1996). In addition, CRS post-transcriptional regulation of human interleukin-2 gene expression occurs at the level of processing of precursor transcripts, which may be linked to the presence of a protein. Gerez, et al. J. Biol Chem. 270:19569 (1995).
Thus, therapeutic agents can be designed that interfere with helicase activity or RNA processing that is associated with the disease state.
The isoxazole derivative leflunomide shows anti-inflammatory and immunosuppressive properties without causing damage to the existing functions of the immune system (HWA486 (leflunomide); R. R. Bartlett, G. Campion, P. Musikic, T. Zielinski, H. U. Schorlemmer In: A. L. Lewis and D. E. Furst (editors), Nonsteroidal Anti-inflammatory Drugs, Mechanisms and Clinical Uses (Dekker: New York, 1994); C. C. A. Kxc3xcchle, G. H. Thoenes, K. H. Langer, H. U. Schorlemmer, R. R. Bartlett, R. Schleyerbach, Transplant Proc. 1991, 23:1083-6; T. Zielinski, H. J. Mxc3xcller, R. R. Bartlett, Agents Action 1993, 38:C80-2). Many activities, such as the modification of cell activation, proliferation, differentiation and cell cooperation, which can be observed in autoimmune diseases, are modulated by leflunomide or its active metabolite, A77 1726. 
Studies on the molecular mechanism of action of this active compound point to an influence on the pyrimidine metabolism. Because leflunomide is very rapidly converted in the body into A77 1726, in this specification, leflunomide and A77 1726 are used interchangeably. Thus, both xe2x80x9cleflunomide resistancexe2x80x9d and xe2x80x9cA77 1726 resistancexe2x80x9d are used to designate the same condition.
Pyrimidine and purine nucleotides play a key part in biological processes. As structural units of DNA and RNA, they are thus carriers of genetic information. The biosynthesis of the pyrimidines comprises the irreversible oxidation of dihydroorotate to orotate, which is catalyzed by the enzyme dihydroorotate dehydrogenase (DHODH). Altogether, six enzymes are needed for the de nova synthesis of uridine monophosphate (UMP). UMP plays a key part in the synthesis of the other pyrimidines, cytidine and thymidine. The inhibition of DHODH thus leads to an inhibition of pyrimidine de novo synthesis. Particularly affected are immune cells, which have a very high need for nucleotides, but can only cover a little of this by side routes (salvage pathway). Binding studies with radiolabeled leflunomide analogs identified the enzyme DHODH as a possible site of action of A77 1726 and thus the inhibition of DHODH by leflunomide is an important starting point for the elucidation of the observed immunomodulating activities. Williamson, et al. J. Biol. Chem. 270:22467-22472 (1995).
In one embodiment, the invention provides an isolated DNA sequence encoding a DEAH-box leflunomide-resistant protein. The invention also provides such a DNA sequence wherein said protein has a molecular weight of 135 kilodaltons. The invention also provides such a DNA sequence, wherein said protein has a molecular weight of about 135 kilodaltons.
In another embodiment, the invention provides an isolated DNA sequence as set forth in SEQUENCE ID NO. 15 (FIG. 8) and an isolated DNA sequence as set forth in SEQUENCE ID NO. 17 (FIG. 9). In other embodiments, the invention provides a homolog of the DNA sequence of SEQ. ID. NO. 15 and a homolog of the DNA sequence of SEQ. ID NO. 17.
In another embodiment, the invention provides isolated DNA sequences encoding the amino acid sequence of SEQUENCE ID NO. 16 (FIG. 8) and encoding the amino acid sequence of SEQUENCE ID NO. 18 (FIG. 9).
In yet another embodiment, the invention provides an isolated DNA sequence that encodes a DEAH-box protein having one or more of the following characteristics:
(a) the first homology domain (APTase A, Domain I) is located more than 650 amino acids from the N-terminus of said protein; (b) the N-terminus of said protein contains serine/arginine domains; (c) domain IV of said protein has the sequence FMP; (d) the distance between domains IV and V of said protein is 74 amino acids or less; and (e) domain VI of said protein has the sequence QRSGRXGR.
The invention also provides an expression vector comprising a DNA sequence according to the invention. The invention further provides a host comprising such an expression vector. The invention also provides an antisense expression vector comprising a DNA according to the invention, wherein said DNA sequence is inserted in reverse orientation into said vector.
In another embodiment, the invention provides an isolated leflunomide-resistant DEAH-box protein. The invention also provides such a protein wherein said protein has a molecular weight of 135 kilodaltons. The invention further provides such a protein wherein said protein has a molecular weight of about 135 kilodaltons. The invention also provides a mammalian protein, a protein isolated from a cell line derived from the murine cell line A20.2J and a human protein.
In yet other embodiments of the invention there is provided a protein comprising the amino acid sequence of SEQUENCE ID NO. 16 (FIG. 8), or a fragment thereof, or the amino acid sequence of SEQUENCE ID NO. 18 (FIG. 9), or a fragment thereof.
In yet another embodiment, the invention provides an isolated DEAH-box protein having one or more of the following characteristics: (a) the first homology domain (APTase A, Domain I) is located more than 650 amino acids from the N-terminus of said protein; (b) the N-terminus of said protein contains serine/arginine domains; (c) domain IV of said protein has the sequence FMP; (d) the distance between domains IV and V of said protein is 74 amino acids or less; and (e) domain VI of said protein has the sequence QRSGRXGR.
In another embodiment, the invention provides a process for the preparation of a DEAH-box leflunomide-resistant protein, wherein said process comprises:
(a) culturing a host cell comprising a vector encoding a DEAH-box leflunomide-resistant protein and
(b) isolating said protein from the cell of step (a).
In yet another embodiment, the invention provides an xe2x80x9cidentifyingxe2x80x9d method for identifying a substance having one or more of the following properties: anticarcinogenic, anti-atherosclerotic, immunosuppressive, antiinflammatory, antiviral, antifungal or antibacterial, comprising:
(a) crystallizing a protein according to the invention;
(b) determining the three-dimensional structure said protein; and
(c) identifying said substance using molecular modeling techniques, wherein said substanceaffects the ability of said protein to bind ATP or nucleic acid.
The invention further provides such an identifying method wherein the method comprises the additional step of determining the ability of the identified substance to modulate the helicase activity of said DEAH-box leflunomide-resistant protein. The invention also provides such an identifying method comprising the additional step of determining the ability of the identified substance to modulate the ATPase activity of said protein. Finally the invention provides such an identifying method comprising the additional step of determining the ability of the identified substance to modulate the splicing activity of said protein. In another embodiment, the invention provides a substance identified using any of the foregoing methods.
In yet another embodiment, the invention provides a therapeutic method for the treatment of a disorder selected from the group consisting of Alzheimer""s disease, cancer, rheumatism, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases, autoimmune disorders, diabetes or organ transplant rejection, comprising administration of a pharmaceutically effective amount of a substance identified using the above-mentioned method to a patient in need of such treatment.
The invention further provides an xe2x80x9cidentifyingxe2x80x9d method for identifying a substance that will modulate the helicase activity of a protein according to the invention, comprising the additional steps of:
(a) transforming a non-leflunomide-resistant cell with a DNA sequence encoding a DEAH-box protein which binds nucleic acid and ATP, and which has helicase activity and ATPase activity, wherein the level of expression of said protein is significantly higher in a leflunomide-resistant cell than in a non-leflunomide-resistant cell, wherein said transformed cell is rendered resistant to leflunomide;
(b) culturing the cells in the presence of a high level of leflunomide;
(c) determining the ability of said substance to make the cells of step (b) non-leflunomide-resistant, wherein a substance that makes said cells non-leflunomide-resistant modulates the helicase activity of said protein.
In another embodiment, the invention provides a method for isolation of RNA that binds specifically to a protein according to the invention, comprising:
(a) binding said protein or a fragment thereof to an affinity matrix;
(b) mixing an RNA sample to the matrix of step (a); and
(c) determining which RNA is specifically bound to said matrix.
The invention also provides such a method comprising the additional step of amplifying the RNA bound to said matrix by using the PCR technique. The invention also provides such a method, wherein said RNA of step (c) is subjected to sequence analysis.
Finally, in another embodiment, the invention provides a method for selecting a cell that contain heterologous DNA comprising:
(a) transforming cells with a vector comprising a DNA sequence encoding a DEAH-box leflunomide-resistant protein;
(b) growing said cells in the presence of a high level of leflunomide; and
(c) selecting a cell that will grow in the presence of said high level of leflunomide;
wherein said cell of step (c) contains said heterologous DNA.